SCIE.16.166 - CELLULAR SENESCENCE AND INFLAMMATORY PROGRAMS ARE UNINTENDED CONSEQUENCES OF CRISPR-CAS9 GENE EDITING IN HEMATOPOIETIC STEM AND PROGENITORS CELLS

TOPIC:
Genetic immune disease
AUTORI:
Conti A. (Milan) , De Marco R. (Milan) , Tavella T. (Milan) , Midena F. (Milan) , Ferrari S. (Milan) , Beretta S. (Milan) , Brombin C. (Milano) , Merelli I. (Segrate Milano) , Naldini L. (Milan) , Di Micco R. (Milan)
Abstract:
Gene editing (GE) in hematopoietic stem and progenitor cells (HSPC) is a revolutionary site-specific gene correction strategy for a plethora of immune-hematological diseases. Despite the rapid development of advanced GE-based therapies, a few challenges remain to be faced to improve GE efficiency and the reduced repopulating potential upon transplantation. We previously showed that the combination of nuclease-induced Double Strand Break with DNA repair template for Homology Directed Repair (HDR) delivered by AAV6 caused cumulative activation of the p53-mediated DNA Damage Response (DDR) pathway constraining HSPC proliferation and yield, suggesting that DDR-related cellular programs may inadvertently contribute to HSPC dysfunctions upon gene editing. Protracted DDR signaling has been causatively linked to the establishment of cellular senescence, a condition in which cells, despite being still alive, are unable to further proliferate. By integrating transcriptional analysis (up to the single cell level) with innovative imaging-based cellular assays we reported induction of cellular senescence markers (p16 and Senescence-Associated β-Galactosidase) and pro-inflammatory programs across HSPC subtypes and in vivo in the human graft, specifically in cells that have undergone HDR. Consistently, we found open chromatin at promoters of several senescence-gene categories and inflammatory genes of the IL1 axis (an upstream mediator of DDR-dependent inflammation) and NF-kB pathway (a key regulator of inflammatory genes upon several stressors). Moreover, transcriptional activation of inflammatory cytokines in edited HSPC was dependent on the apical DDR kinase ATM and partly mitigated by transient administration of a p53 dominant negative form (GSE56). In this context, temporary inhibition of IL1 and NF-kB pathways at the time of GE resulted in increased clonogenicity of edited HSPC in vitro and ameliorated long-term hematopoietic reconstitution in xenotranspanted mice. We also reported a decrease in senescence markers in both cord blood and mobilized peripheral blood-derived HSPC upon GE. By a barcoding-based strategy, we performed in vivo clonal tracking of HDR-edited HSPC revealing that IL1 inhibition improved polyclonal reconstitution and better preserved self-renewal and multi-potency of individual edited HSPC. Our findings define senescence and inflammatory programs as long-term consequences of CRISPR-Cas9 engineered human HSPC and pave the way for the development of novel strategies based on senescence modulation and anti-inflammatory molecules to overcome adverse cellular responses for efficient HSPC-based clinical applications.
Abstract per il pubblico laico:
Studiare l'impatto delle strategie di ingegneria genetica sulla biologia delle staminali del sangue. Le cellule staminali ematopoietiche hanno l'enorme potenziale di dar vita a tutte le cellule mature del sangue. Purtroppo, con l'avanzare dell'età, le cellule staminali invecchiano e non riescono più a garantire l'omeostasi tissutale. In maniera simile, le cellule staminali sottoposte allo stress del trapianto o manipolate in coltura per approcci di terapia genica possono accidentalmente attivare delle risposte cellulari che ne limitano la ricostituzione ematopoietica, con segni di invecchiamento prematuro. La nostra ricerca ha lo scopo di identificare i meccanismi molecolari che causano la ridotta funzionalità delle staminali ematopoietiche durante l'invecchiamento fisiologico e in risposta alla manipolazione genica, con il fine ultimo di sviluppare delle nuove strategie di "ringiovanimento" delle stesse per applicazioni di terapia genica e cellulare più ampie e sicure.
Disease Name:
Dyskeratosis Congenita
Nome malattia:
Dyskeratosis Congenita